The peptide hormone, relaxin, is a pregnancy-associated polypeptide, which is present in some analogous form in most mammalian species, including human, porcine, equine, canine, rodent and feline. The relaxin molecule is processed in the body as a single prepropolypeptide containing a signal peptide, followed by a B chain, which is connected by a connecting peptide (C peptide) to an A chain. During processing the signal peptide and the C peptide are removed from the relaxin molecule. Thus the biologically active relaxin molecule present in the serum is formed of the A and B chains paired by disulfide bonds into an appropriately folded and active polypeptide.
Immunoreactivity of relaxin requires native conformation [B. E. Kemp et al., Vitamins and Hormones, 41:79-115 (1984)]. When the disulfide bonds of the 6300 dalton di-peptide are reduced, immunoreactivity is lost. Similarly, it is difficult to synthesize an active or immunoreactive relaxin due to the need to properly align the two interchain and one intrachain disulfides, even when a portion of the B-chain carboxy terminus is deleted to improve its solubility properties [B. E. Kemp et al., cited above].
The amino acid sequences of the prepropolypeptide of relaxin in porcine, rat and human are known and published in, for example, Bruce E. Kemp and Hugh D. Nile, Vitamins and Hormones, Vol. 41, pp. 79-115 (1984). A variety of mammalian species of relaxin and its prepropolypeptide forms demonstrate highly conserved genetic regions. The C peptide of relaxin is a larger sequence than the relaxin polypeptide itself. Because the C peptide is not itself a hormone, it is not as sensitive to degradation in the excretory or other systems.
Relaxin has been inconsistently reported in some instances as detectable in urine and in other instances as not detectable. See, e.g., West German patent application No. 3236267, published Apr. 5, 1984 which discusses a purification technique for the recovery of relaxin from urine; and U. Fuchs et al., "Presence of Immunoreactive Relaxin in Human Female Urine", Arch. Gynecol. 237 (Suppl.): 383 (1985). Relaxin may be denatured or degraded before being excreted and, as such, lose its immunoreactivity. Without these disulfide bonds it is probable that the relaxin molecule loses its antigenicity, if it is in fact present in urine.
The presence of relaxin in serum is presently the only good early indicator of true pregnancy in dogs [B. G. Steinetz et al., Biol. Reproduct., 37:719-725 (1987) (Steinetz I); and B. G. Steinetz et al., Am. J. Vet. Res., 50:68-71 (1989) (Steinetz II)]. Currently, canine serum relaxin is measured with a radioimmunoassay (RIA) over a three day protocol [E. M. O'Byrne et al., Proc. Soc. Exo. Biol. and Med., 152:272-276 (1976); and O. D. Sherwood et al., Endocrinology, 96:1106-1113 (1975)]. Pregnancy is indicated upon observation of detectable amounts of relaxin, usually observed in pregnant dogs around the twenty-eighth day after the luteinizing hormone (LH) peak (day zero). The serum relaxin RIA test, however, still requires the help of a veterinarian or trained technician to draw blood samples.
Problems remain with utilizing this assay as a convenient method of pregnancy detection in animals. For example, serum diagnosis of pregnancy in animals is simply impractical as a means for the animals'owner or breeder to routinely practice. There is a need for reliable pregnancy detection methods because certain animals, particularly dogs, often show overt symptoms of pseudopregnancy indistinguishable from the symptoms of actual pregnancy.
Thus there remains a need in the art for convenient means for detection of the condition of pregnancy in animals, particularly for domestic animals such as dogs and cats.